title: Effect of calcitonin gene-related peptide on nitric oxide production in osteoblasts: an experimental study.
abstract: The aim of this study was to investigate the in vitro effects and regulatory mechanism of CGRP (calcitonin gene-related peptide) on NO (nitric oxide) production in osteoblasts. MOB (primary human mandibular osteoblasts) and osteoblast-like cells (MG-63) were either cultured with CGRP or co-incubated with inhibitors targeting eNOS (endothelial nitric oxide synthase), iNOS (inducible nitric oxide synthase), nNOS (neuronal nitric oxide synthase) and [Ca2+]i (intracellular Ca2+). The NO concentration in cell culture supernatants was measured during the first 24 h using the Griess test; cellular NO was marked with the fluorescent marker DAF-FM, DA (3-amino, 4-aminomethyl-2',7'-difluorescein; diacetate) and measured by fluorescence microscopy from 1 to 4 h after treatment. eNOS and iNOS mRNA expression levels were measured by quantitative RT-PCR during the first 24 h after treatment. CGRP-induced NO production in the supernatants was high between 1 to 12 h, while cellular NO was highest between 1 to 2 h after treatment and returned to basal levels by 3 h. Both in MG-63 cells and MOBs, the most effective CGRP concentration was 10 nM with a peak time of 1 h. CGRP-induced NO production decreased when eNOS activity was inhibited or when voltage-dependent L-type Ca2+ channels were blocked at 4 h. CGRP was not able to induce changes in iNOS or eNOS mRNA levels and had no effect on the cytokine-induced increase of iNOS expression. Our results suggest that CGRP transiently induces NO production in osteoblasts by elevating intracellular Ca2+ to stimulate the activity of eNOS in vitro.
authors.affiliation: Department of Oral and Maxillofacial Surgery, Xingqiao Hospital, Third Military Medical University, Chongqing, Peoples Republic of China.
authors.forename: Li
authors.initials: L
authors.lastname: Yan
authors.forename: Tan
authors.initials: T
authors.lastname: Yinghui
authors.forename: Yang
authors.initials: Y
authors.lastname: Bo
authors.forename: Zhang
authors.initials: Z
authors.lastname: Gang
authors.forename: Xiao
authors.initials: X
authors.lastname: Xian
authors.forename: Zheng
authors.initials: Z
authors.lastname: Lu
mesh_terms: D015740:Calcitonin Gene-Related Peptide / Q000378:metabolism*; D002118:Calcium / Q000037:antagonists & inhibitors; D045744:Cell Line, Tumor; D016207:Cytokines / Q000494:pharmacology; D006801:Humans; D008856:Microscopy, Fluorescence; D009569:Nitric Oxide / Q000096:biosynthesis*; D052248:Nitric Oxide Synthase Type I / Q000037:antagonists & inhibitors; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors; D010006:Osteoblasts / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D016207:Cytokines; D012333:RNA, Messenger; D009569:Nitric Oxide; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D015740:Calcitonin Gene-Related Peptide; D002118:Calcium